Objectives: The objective of this study was to determine the effects of antimicrobial photodynamic therapy (aPDT) with indocyanine green (ICG) and toluidine blue (TB) on protease activity (matrix-bound cathepsin K and matrix metalloproteinase (MMP) and dentin bond strength. Methods: Caries-free human third molars were assigned to five groups: 1—control group, 2—application of ICG with activation using an 810 nm diode (aPDT), 3—application of ICG, 4—application of TB with activation using a 660 nm diode (aPDT), and 5—application of TB. For the enzymatic investigation, dentin beams were incubated for either 3 days or 3 weeks. Aliquots of the incubation media were analyzed by ELISA for CTX (C-terminal cross-linked telopeptide of type I Collagen) and ICTP (cross-linked carboxy-terminal telopeptide of type I collagen). For microtensile bond strength testing (μTBS), composite resins were layered onto the tooth surface; the samples were then subjected to μTBS. Kruskall–Wallis and Mann–Whitney U tests were applied for statistical analysis of CTX and ICTP, one way-ANOVA and Tukey's test were applied for statistical analysis of μTBS. Results: Pretreating the dentin matrices with aPDT decreased the endogenous protease activity. ICG with laser activation resulted in the highest μTBS. Therefore, aPDT should be considered as a treatment method because it can reduce MMP-mediated dentin degradation and increase the μTBS. Significance: Inhibiting endogenous protease activity improves the stability of the dentin–adhesive bond and the durability of the bond strength.